Peptides having anti-cancer and anti-inflammatory activity

ABSTRACT

A pentapeptide is disclosed having the generic formula A-A-A-B-C (SEQ ID NO:2) wherein A is a non-polar amino acid, B is a polar amino acid, and C is a charged amino acid. In a preferred embodiment the peptide has the sequence A-Pro-Pro-B-C (SEQ ID NO:3), and in a further preferred embodiment has the sequence of leucine-proline-proline-serine-argenine (SEQ ID NO:1). In a most preferred embodiment, the peptide comprises at least one D-amino acid. The peptide can be extracted from the epidermis of sea cucumbers. The peptides of the present invention are useful for inhibition of tumor progression and/or inflammation in a mammal by administration from 1 milligram per kilogram body weight to 5000 milligrams per kilogram body weight. The peptide can be administered in conjunction with any suitable carriers or excipients as are known those skilled in the arts via oral delivery forms, such as in capsules, drinks, powders, rectally via suppositories, or other suitable means.

This application is a 371 application of PCT/US99/27289, filed on Nov.18, 1999, and claims priority to Provisional Application 60/109,139,filed Nov. 18, 1998, and to Provisional Application 60/157,078, filed onOct. 1, 1999.

TECHNICAL FIELD

The present invention relates to the fields of cancer treatment,inflammatory disease, and peptide chemistry.

BACKGROUND

The amino acid sequence Leu-pro-pro-ser-arg (SEQ ID NO:1) is a knownimmunostimulant and is described in papers by W. Weigle and is thesubject of a US Patent. Briefly, the sequence of the present inventionhas been examined in U.S. Pat. No. 4,683,221 and claims were made forthe stimulation of lymphocyte proliferation in mammals. U.S. Pat. No.4,683,221 is included herein by reference, and contains descriptions ofthe biological effects of the SEQ. ID. 1 with regards to B cells, B celldifferentiation and macrophages in vivo and in vitro experiments.Nowhere in the Weigle patent is mention of angiogenesis inhibition northe connection between lymphocyte activiation and the inhibition ofhuman neoplasms nor inflammation inhibition. Based on the work describedin the Weigle patent, one would anticipate that the strongimmuno-stimulating effect of the peptide leu-pro-pro-ser-arg (SEQ IDNO:1) would lead to increased inflammatory response.

Also, the Weigle patent does not describe any production methods of thesubject peptide as being available from sea cucumber epithelial or othertissue.

Angiogenesis is the generation of new blood vessels into a tissue ororgan. Under normal physiological conditions, humans or animals onlyundergo angiogenesis in very specific restricted situations. Forexample, angiogenesis is normally observed in wound healing, fetal andembryonal development and formation of the corpus luteum, endometriumand placenta. The control of angiogenesis is a highly regulated systemof angiogenic stimulators and inhibitors. The control of angiogenesishas been found to be altered in certain disease states and, in manycases, the pathological damage associated with the disease is related tothe uncontrolled angiogenesis.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel. In the disease state,prevention of angiogenesis could avert the damage caused by the invasionof the new microvascular system. Persistent, unregulated angiogenesisoccurs in a multiplicity of disease states, tumor metastasis andabnormal growth by endothelial cells and supports the pathologicaldamage seen in these conditions. The diverse pathological states createddue to unregulated angiogenesis have been grouped together as angiogenicdependent or angiogenic associated diseases. Therapies directed atcontrol of the angiogenic processes could lead to the abrogation ormitigation of these diseases.

One example of a disease mediated by angiogenesis is ocular neovasculardisease. This disease is characterized by invasion of new blood vesselsinto the structures of the eye such as the retina or cornea. It is themost common cause of blindness and is involved in approximately twentyeye diseases. In age-related macular degeneration, the associated visualproblems are caused by an ingrowth of chorioidal capillaries throughdefects in Bruch's membrane with proliferation of fibrovascular tissuebeneath the retinal pigment epithelium. Angiogenic damage is alsoassociated with diabetic retinopathy, retinopathy of prematurity,corneal graft rejection, neovascular glaucoma and retrolentalfibroplasia. Other diseases associated with corneal neovascularizationinclude, but are not limited to, epidemic keratoconjunctivitis, VitaminA deficiency, contact lens overwear, atopic keratitis, superior limbickeratitis, pterygium keratitis sicca, sjogrens, acne rosacea,phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration,chemical burns, bacterial ulcers, fungal ulcers, Herpes simplexinfections, Herpes zoster infections, protozoan infections, Kaposisarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginalkeratolysis, rheumatoid arthritis, systemic lupus, polyarteritis,trauma, Wegeners sarcoidosis, Scleritis, Steven's Johnson disease,periphigoid radial keratotomy, and corneal graph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagetsdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, Eales disease,Bechets disease, infections causing a retinitis or choroiditis, presumedocular histoplasmosis, Bests disease, myopia, optic pits, Stargartsdisease, pars planitis, chronic retinal detachment, hyperviscositysyndromes, toxoplasmosis, trauma and post-laser complications. Otherdiseases include, but are not limited to, diseases associated withrubeosis (neovasculariation of the angle) and diseases caused by theabnormal proliferation of fibrovascular or fibrous tissue including allforms of proliferative vitreoretinopathy.

Another disease in which angiogenesis is believed to be involved isrheumatoid arthritis. The blood vessels in the synovial lining of thejoints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis.

Factors associated with angiogenesis may also have a role inosteoarthritis. The activation of the chondrocytes by angiogenic-relatedfactors contributes to the destruction of the joint. At a later stage,the angiogenic factors would promote new bone formation. Therapeuticintervention that prevents the bone destruction could halt the progressof the disease and provide relief for persons suffering with arthritis.

Chronic inflammation may also involve pathological angiogenesis. Suchdisease states as ulcerative colitis and Crohn's disease showhistological changes with the ingrowth of new blood vessels into theinflamed tissues. Bartonellosis, a bacterial infection found in SouthAmerica, can result in a chronic stage that is characterized byproliferation of vascular endothelial cells. Another pathological roleassociated with angiogenesis is found in atherosclerosis. The plaquesformed within the lumen of blood vessels have been shown to haveangiogenic stimulatory activity.

One of the most frequent angiogenic diseases of childhood is thehemangioma. In most cases, the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, the hemangiomatoses, have a highmortality rate. Therapy-resistant hemangiomas exist that cannot betreated with therapeutics currently in use.

Angiogenesis is also responsible for damage found in hereditary diseasessuch as Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumors of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epistaxis(nosebleeds) or gastrointestinal bleeding and sometimes with pulmonaryor hepatic arteriovenous fistula.

Angiogenesis is prominent in solid tumor formation and metastasis.Angiogenic factors have been found associated with several solid tumorssuch as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma,and osteosarcoma. A tumor cannot expand without a blood supply toprovide nutrients and remove cellular wastes. Tumors in whichangiogenesis is important include solid tumors, and benign tumors suchas acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.Prevention of angiogenesis could halt the growth of these tumors and theresultant damage to the animal due to the presence of the tumor.

It should be noted that angiogenesis has been associated with blood-borntumors such as leukemias, any of various acute or chronic neoplasticdiseases of the bone arrow in which unrestrained proliferation of whiteblond cells occurs, usually accompanied by anemia, irpaired bloodclotting, and enlargement of the lymph nodes, liver, and spleen. It isbelieved that angiogenesis plays a role in the abnormalities in the bonemarrow that give rise to leukemia-like tumors.

Angiogenesis is important in two stages of tumor metastasis. The firststage where angiogenesis stimulation is important is in thevascularization of the tumor which allows minor cells to enter the bloodstream and to circulate throughout the body. After the tumor cells haveleft the primary site, and have settled into the secondary, metastasissite, angiogenesis must occur before the new tumor can grow and expand.Therefore, prevention of angiogenesis could lead to the prevention ofmetastasis of tumors and possibly contain the neoplastic growth at theprimary site.

Knowledge of the role of angiogenesis in the maintenance and metastasisof tumors has led to a prognostic indicator for breast cancer. Theamount of neovascularization found in the primary tumor was determinedby counting the microvessel density in the area of the most intenseneovascularization in invasive breast carcinoma. A high level ofmicrovessel density was found to correlate with tumor recurrence.Control of angiogenesis by therapeutic means could possibly lead tocessation of the recurrence of the tumors.

Angiogenesis is also involved in normal physiological processes such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation or to prevent implantation by the blastula.

In wound healing, excessive repair or fibroplasia can be a detrimentalside effect of surgical procedures and may be caused or exacerbated byangiogenesis. Adhesions are a frequent complication of surgery and leadto problems such as small bowel obstruction.

Several kinds of compounds have been used to prevent angiogenesis.Taylor et al. have used protamine to inhibit angiogenesis, see Taylor etal., Nature 297:307 (1982). The toxicity of protamine limits itspractical use as a therapeutic. Folkman et al. have disclosed the use ofheparin and steroids to control angiogenesis. See Folkman et al.,Science 221:719 (1983) and U.S. Pat. Nos. 5,001,116 and 4,994,443.Steroids, such as tetrahydrocortisol, which lack gluco and mineralcorticoid activity, have been found to be angiogenic inhibitors.

Other factors found endogenously in animals, such as a 4 kDaglycoprotein from bovine vitreous humor and a cartilage derived factor,have been used to inhibit angiogenesis. Cellular factors such asinterferon inhibit angiogenesis. For example, interferon .alpha. orhuman interferon .beta. has been shown to inhibit tumor-inducedangiogenesis in mouse dermis stimulated by human neoplastic cells.Interferon .beta. is also a potent inhibitor of angiogenesis induced byallogeneic spleen cells. See Sidky et al., Cancer Research 47:5155-5161(1987). Human recombinant .alpha. interferon (alpha/A) was reported tobe successfully used in the treatment of pulmonary hemangiomatosis, anangiogenesis-induced disease. See White et al., New England J. Med.320:1197-1200 (1989).

Other agents which have been used to inhibit angiogenesis includeascorbic acid ethers and related compounds. See Japanese Kokai TokkyoKoho No. 58-131978. Sulfated polysaccharide DS 4152 also showsangiogenic inhibition. See Japanese Kokai Tokkyo Koho No. 63-119500. Afungal product, fumagillin, is a potent angiostatic agent in vitro. Thecompound is toxic in vivo, but a synthetic derivative, AGM 12470, hasbeen used in vivo to treat collagen II arthritis. Fumagillin andO-substituted fumagillin derivatives are disclosed in EPO PublicationNos. 0325199A2 and 0357061A1.

The above compounds are either topical or injectable therapeutics.Therefore, there are drawbacks to their use as a general angiogenicinhibitor and lack adequate potency. For example, in prevention ofexcessive wound healing, surgery on internal body organs involvesincisions in various structures contained within the body cavities.These wounds are not accessible to local applications of angiogenicinhibitors. Local delivery systems also involve frequent dressings whichare impracticable for internal wounds, and increase the risk ofinfection or damage to delicate granulation tissue for surface wounds.

Thus, a method and composition are needed that are capable of inhibitingangiogenesis and which are easily administered. A simple and efficaciousmethod of treatment would be through the oral route. If an angiogenicinhibitor could be given by an oral route, the many kinds of diseasesdiscussed above, and other angiogenic dependent pathologies, could betreated easily. The optimal dosage could be distributed in a form thatthe patient could self-administer.

Inflammation is a major contributor to many diseases and as such,methods and compositions of matter have been sought to mitigate thedetrimental effects of inappropriate activation.

Dermal inflammation is partially mediated via the conversion ofphospholipids to either endoperoxides and consequently prostaglandinsvia cyclooxygenase and 5-HETE's, or consequently leukotrienes vialipoxygenase (Kraghball and Voorhees. 1985. Curr. Probl. Derm. 13:1-10).Inhibition of either or both of these pathways is the means by whichnon-steroidal anti-inflammatory agents prevent an inflammatory response.Anti-inflammatory steroids act by inhibiting the release of arachidonicacid which can then be converted via either pathway to mediators ofinflammation (Blackwell et al. 1980 Nature 287:147-149). It has beenproposed that cytokines also mediate the inflammatory process and abetter, or at least, equivalent inhibition of the inflammatory responsemay be achieved by inhibiting either cytokine production or theinhibition of the interaction of cytokines with their receptors on thecell surface of the inflammatory cell infiltrate. There have beenseveral peptides isolated from sea cucumber of various species(Bizenheide, R., Tamori, Motokawa, et al. “Peptides ControllingStiffness of Connective Tissue in Sea Cucumbers.” Bio. Bull 194:253-259.June 1998), but none are reported to have the sequence of the peptidesof the present invention, nor do any peptides that has as its activity,the ability to inhibit an inflammatory response in a mammal. As apotentiator of a mammalian immune system response by way of activatinglymphocytes and B cells and the associated cascades of immune-systemprotection, the peptide of the present invention may exert itsanti-inflammatory and anti-cancer effects by an up-regulation of themammalian immune system that is then able to destroy cancer cellsdirectly or competitively bind to Fc receptors, thus mitigating theirbiological effects.

SUMMARY OF THE INVENTION

The present invention relates to a peptide, defined by the genericformula A-A-A-B-C (SEQ ID NO:2), wherein A is a non-polar amino acid, Bis a polar amino acid and C is a charged amino acid. A sub-group ofthese peptides can be described by the formula A-Pro-Pro-B-C (SEQ IDNO:3), wherein A, B,& C have the meanings defined above. A specificembodiment of the peptide comprises the sequence LEU-PRO-PRO-SER-ARG(SEQ. ID NO: 1), which and is a fragment of variousimmunogammaglobulins, and especially of IgG, and described in U.S. Pat.No. 4,683,221 by William Weigle, et al as a portion of the Fc region ofIgG. This peptide was initially isolated from the epidermis of the seacucumber Cucumaria frondosa, and has both anti-cancer and potentanti-inflammatory activities. In a preferred embodiment the peptidecomprises at least one D-amino acid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the mass spectroscopy spectrum of the purified peptide.

FIG. 2 shows the mass spectrum of a synthetic preparation of the peptideof SEQ. ID NO. 1.

FIG. 3 shows an HPLC trace indicating the purity of the syntheticpeptide preparation.

FIG. 4 shows the anti-inflammatory response of the peptide AdjuvantInduced Arthritis Model.

DETAILED DESCRIPTION

The present invention relates in part to the anti-cancer activity ofpeptides and peptide derivatives related to defense mechanisms presenton the epithelia of echinoderms and especially, of the sea cucumber,Cucumaria frondosa. The invention includes both pharmaceuticalcompositions comprising said peptides, and methods for inhibiting tumorprogression utilizing said peptides. The invention is based in part onthe discovery that the pentapeptide LEU-PRO-PRO-SER-ARG (SEQ. ID NO: 1)and analogues have potent anti-cancer activity as evidenced by activityin various screening assays such as those evaluating tumor necrosisfactor (TNF-α) and interleukin-2 (IL-2) response, the inhibition ofcancer as evidenced in a murine model of Sarcoma 180, the inhibition ofangiogenesis as evidenced in the CAM assay of neovascularization. Thesetwo activities are correlated to anti-cancer effect. (See, e.g., “T-CellActivation as Cancer Treatment,” Conrad Notes(http://www./meds.com/conrad/ecco/nielsen.html) (1999) (presented at theEuropean Cancer Conference (ECCO 9), Sep. 14-18, 1997).

Contrary to what would be expected from the teachings of the Weiglepatent, the peptides of the present invention also may be used toinhibit inflammation in a tissue of a subject in need of such treatmentsuffering from a disease or disorder in which either acute or chronicinflammatory cell influx occurs. While not wishing to be bound bytheory, this unexpected effect is believed to be due to the presence ofone or more D-conformation amino acids in the sequence of the peptideisolated from sea cucumber, which alters the competitive binding of thispeptide with receptor sites. The subject may be a human or non-humansubject. Specific conditions in which peptides of the invention may havetherapeutic value would include situations in which undesirable immuneresponse has occurred, including, but not limited to, autoimmunediseases such as insulin-dependent diabetes, Goodpasture's syndrome,pemphigus and pemphigoid, primary biliary cirrhosis, ulcerative colitis,rheumatoid arthritis, scleroderma, mixed connective tissue disease andlupus erythematosus; graft versus host disease; septic shock;reperfusion injury (including injury subsequent to myocardial orcerebral infarction atherosclerosis; asthma, psoriasis and inflammatorylung disease. In preferred specific embodiments of the invention thepeptide used to inhibit inflammation is SEQ. NO. 1 (Leu, Pro, Pro, Ser,Arg). This embodiment of the invention is based in part on the discoverythat the peptides have potent anti-inflammatory activity, as evidence bya number of in vivo and in vitro tests, as well as from the realizationthat the tissue of origin of this peptide contains potentanti-inflammatory activity (U.S. Pat. No. 5,770,205).

The peptides of the invention include any peptide, peptide derivative orpeptide analog which comprises either (i) as least three amino acidresidues of SEQ. ID NO: 1, or (ii) a functionally equivalent sequence,or (iii) at least a 4 amino acid sequence which is at least 66%homologous to the corresponding portion of SEQ. ID NO. 1. Analysis ofpreparations of the peptide purified from sea cucumber indicate thepresence of at least one D-amino acid in the sequence.

Peptides of the present invention can be described generically by theformula A-A-A-B-C (SEQ ID NO:2), wherein A is a non-polar amino acid, Bis a polar amino acid and C is a charged amino acid. A sub-group ofthese peptides can be described by the formula A-Pro-Pro-B-C (SEQ IDNO:3), wherein A, B, & C have the meanings defined above. In thepreferred embodiments of the invention, the peptide of the presentinvention, or its derivatives or analogs, comprises the sequenceleucine-proline-proline-serine-arginine. The invention also refers topeptides in which certain residues are substituted by functionallyequivalent amino acids resulting in a silent change. For example, one ormore amino acids residues within the sequence can be substituted byanother amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the nonpolar amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan and methionine. The polar neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. In a preferred embodiment, thepeptide comprises at least one D-amino acid.

The peptides of the present invention may be prepared by any methodknown in the art. For example, and not by way of limitation, thepeptides may be synthesized: (i) by cleavage from a large peptide; (ii)by recombinant DNA expression methods; and (iii) by chemical synthesis,including solid phase techniques as described by Barany and Merrifield(1980 in “The Peptides: Vol. 2.” Gross and Meienhofer, Eds., AcademicPress, N.Y.).

The peptides of the invention may be administered by any suitable andaccepted route of drug administration, including intravenous,subcutaneous, intradermal, intranasal, inhalation, intramuscular,intraocular, intraperitoneal injection, peritoneal lavage intranasal,inhalation, intramuscular, intraocular, intraperitoneal injection,peritoneal lavage, cardiac puncture, cardiac catheter injection, oral,intrathecal or intraventricular injection, spinal column or cranialcavity injection, vaginal or rectal dermal patch or topical ointment,and may be comprised in any suitable pharmaceutical carrier, includingaqueous solution, micrbcapsules, liposomes, or via a sustained-releaseimplant, including hydrophilic or hydrophobic carrier-based implants.

The compositions of the invention may be presented in pharmaceuticaldosage forms normally used, depending on whether the composition has tobe swallowed, injected or applied to the skin or mucosae.

For injection, the composition may take the form of an aqueous or oilylotion or the form of a serum.

For swallowing, the composition may take the form of capsules, syrupgranules or tablets, or it can be incorporated appropriately intofood-stuffs as a supplement to the food composition.

In the topical compositions of the invention, the peptide can preferablybe used in an amount ranging from 1% to 100% by weight relative to thetotal weight of the composition, and especially in an amount rangingfrom 1% to 20% by weight relative to the total weight of thecomposition.

For topical application, the composition may take the form, inparticular, of aqueous or oily solutions or of dispersions of the lotionor serum type, of emulsions, of liquid or semi-liquid consistency of themilk type, obtained by dispersing a fatty phase in an aqueous phase(O/W) or vice versa (W/O), or of suspensions or emulsions of softconsistency of the cream or aqueous gel type or which are anhydrous, ofmicroemulsions or alternatively of microcapsules or microparticles, orof vesicular dispersions of the ionic and or nonionic type. Thesecompositions are prepared according to the standard methods.

It may also be used for the scalp in the form of aqueous, alcoholic oraqueous-alcoholic solutions, or in the form of creams, gels, emulsionsor foams, or alternatively in the form of aerosol compositions alsocontaining a propellant agent under pressure.

In a known manner, the pharmaceutical or dermatological composition ofthe invention can also contain adjuvants which are customary in thefields in question, such as hydrophilic or lipophilic gelling agents,preservatives, antioxidants, solvents, perfumes, filler, sunscreenagents, odor absorbers and coloring mater. The amounts of thesedifferent adjuvants are those traditionally used in the cosmetic and/orpharmaceutical field, and are, for example from 0.1% to 10% of the totalweight of the composition. The adjuvants, depending on their nature, maybe introduced into the fatty phase, into the aqueous phase and/or intolipid spherules.

As oils which can be used in the invention, mineral oils, vegetableoils, animal oils, synthetic oils, silicone oils or waxes andfluorinated oils may be mentioned. Fatty alcohols and fatty acids may beadded to these oils. Waxes such as beeswax and carnauba wax or paraffinmay also be used.

As emulsifiers which can be used in the invention, glycerol stearate,polysorbate 60 and the PEG-6/PEG-32/glycol stearate, mixture sold underthe name of Tefose 63 by the company Gattefosse may be mentioned asexamples.

As solvents which can be used in the invention, lower alcohols, inparticular ethanol and isopropanol, and propylene glycol may bementioned.

As hydrophilic gelling agents, carboxyvinyl polymers (carbomer), acryliccopolymers such as acrylate/alkylacrylate copolymers, polyacrylamides,polysaccharides such as hydroxypropylcellulose, clays and natural gumsmay be mentioned, and as lipophilic gelling agents, modified clays suchas bentones, metal salts of fatty acids, such as aluminum stearates andhydrophobic silica may be mentioned.

It is, in addition, possible to introduce hydrophilic active agents suchas proteins or protein hydrolysates, amino acids, polyols, urea, sugars,and sugar derivatives, vitamins, starch, plant extracts such as aloevera and hydroxy acids such as lactic acid or tartaric acid.

It is also possible to introduce lipophilic active agents such asretinol and its derivatives, retinoids such as 13-cis- orall-trans-retinoic acid, tocopherol and its derivatives, essential fattyacids, ceramides, essential oils and salicylic acid and its derivatives.Salicylic, lactic, acetic and the like, acids act, in particular asantiseptics.

Pentapeptides isolated using the isolation method from the sea cucumberdetailed below, or produced from direct peptide synthesis can becombined with pharmaceutical carriers to make novel dosage forms. Thesepeptide based drugs trigger T-cell activation resulting in tumorinhibition. Effective dosages for the treatment of cancer include from 1milligram per kilogram to 5000 milligrams per kilogram body weight ofthe individual in need of treatment.

The peptides of the invention also may be administered at a doseeffective in inhibiting inflammation in the subject as determined usingstandard techniques. “Inhibiting inflammation” should be construed torefer to a significant decrease in the signs and symptoms ofinflammation. For example, but not by way of limitation, symptomaticrelief, in which a patient is rendered subjectively relieved ofdiscomfort, would be considered as satisfactory results of therapy. Incertain specific non-limiting embodiments, the amount of inflammationmay be decreased by about 50%, the ED50 has been estimated to be a dosebetween about 1 and 40 mg/kg. In specific embodiments of the invention,the peptide may be administered to a human patient at a dose of about2.5 mg/kg to about 500 mg/kg. In preferred, specific, non-limitingembodiments of the invention, the dose, administered topically to ahuman patient, may be either about 5 mg/kg, to 100 mg/kg, depending uponwhether the inflammation to be treated is mild, moderate, orsevere/persistent. The dose may be administered at appropriateintervals, e.g. but not limited to, daily, or once, twice, or threetimes a week. In another preferred, specific, non-limiting embodiment ofthe present invention, soft or hard gelatin capsules containingapproximately 500 milligrams of the peptide are administered atapproximately 30 mg/kg of body weight of an animal.

Angiogenesis related pathology in Rheumatoid Arthritis: The developmentof an extensive network of new blood vessels is essential to thedevelopment of the synovitis present in rheumatoid arthritis (Harris,1990; Folkman et al., 1989; Sano et al., 1990). Several local mediatorssuch as platelet derived growth factor (PDGF), TGF-.beta., andfibroblast growth factor (FGF) are likely responsible for the inductionand perpetuation of neovascularization within the synovium. Pannustissue composed of new capillaries and synovial connective tissueinvades and destroys the articular cartilage. The migrating angiogenicvessels themselves produce and secrete increased levels ofmetalloproteinases such as collagenase and stromelysin capable ofdegrading the cartilage matrix (Case et al., 1989). The newly formedvessels are also quite “leaky” with gaps present between themicrovascular endothelial cells. This facilitates the exudation ofplasma proteins into the synovium (which increases swelling), enhancesWBCs movement from the circulation into the pannus tissue (whichincreases inflammation), and leads to the perivascular accumulation ofmononuclear inflammatory cells (Wilder et al., 1991). In summary, theendothelial tissue plays an important role in the development of thisdisease by expressing the necessary surface receptors to allowinflammatory cells to leave the circulation and enter the developingpannus, secreting proteolytic enzymes capable of degrading the cartilagematrix, and proliferating to form the new vessels (angiogenesis)required for the pannus tissue to increase in size and invade adjacenttissues. InflaStatin is contemplated to be injected into the synovium orarticular spaces of joints of Rheumatoid arthritis patients whereby itsanti-angiogenic effects will ameliorate the pathological angiogenesisactivity therein.

Within one embodiment, inhibition of new blood vessel formation may bereadily determined in a variety of assays, including the CAM assaydescribed above.

NEOVASCULAR DISEASES OF THE EYE: As noted above, the present inventionalso provides methods for treating neovascular diseases of the eye,including for example, corneal neovascularization, neovascular glaucoma,proliferative diabetic retinopathy, retrolental fibroblasia and maculardegeneration.

Briefly, corneal neovascularization as a result of injury to theanterior segment is a significant cause of decreased visual acuity andblindness, and a major risk factor for rejection of corneal allografts.As described by Burger et al., Lab, Invest. 48:169-180, 1983, cornealangiogenesis involves three phases: a pre-vascular latent period, activeneovascularization, and vascular maturation and regression. The identityand mechanism of various angiogenic factors, including elements of theinflammatory response, such as leukocytes, platelets, cytokines, andeicosanoids, or unidentified plasma constituents have yet to berevealed.

Currently no clinically satisfactory therapy exists for inhibition ofcorneal neovascularization or regression of existing corneal newvessels. Topical corticosteroids appear to have some clinical utility,presumably by limiting stromal inflammation.

Thus, within one aspect of the present invention methods are providedfor treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of an anti-angiogenic composition (as described above)to the cornea, such that the formation of blood vessels is inhibited.Briefly, the cornea is a tissue which normally lacks blood vessels. Incertain pathological conditions however, capillaries may extend into thecornea from the pericorneal vascular plexus of the limbus. When thecornea becomes vascularized, it also becomes clouded, resulting in adecline in the patient's visual acuity. Visual loss may become completeif the cornea completely opacitates.

Blood vessels can enter the cornea in a variety of patterns and depths,depending upon the process which incites the neovascularization. Thesepatterns have been traditionally defined by ophthalmologists in thefollowing types: pannus trachomatosus, pannus leprosus, pannusphylctenulosus, pannus degenerativus, and glaucomatous pannus. Thecorneal stroma may also be invaded by branches of the anterior ciliaryartery (called interstitial vascularization) which causes severaldistinct clinical lesions: terminal loops, a “brush-like” pattern, anumbel form, a lattice form, interstitial arcades (from episcleralvessels), and aberrant irregular vessels.

A wide variety of disorders can result in corneal neovascularization,including for example, corneal infections (e.g., trachoma, herpessimplex keratitis, leishmaniasis and onchocerciasis), immunologicalprocesses (e.g., graft rejection and Stevens-Johnson's syndrome), alkaliburns, trauma, inflammation (of any cause), toxic and nutritionaldeficiency states, and as a complication of wearing contact lenses.

While the cause of corneal neovascularization may vary, the response ofthe cornea to the insult and the subsequent vascular ingrowth is similarregardless of the cause. Briefly, the location of the injury appears tobe of importance as only those lesions situated within a criticaldistance of the limbus will incite an angiogenic response. This islikely due to the fact that the angiogenic factors responsible foreliciting the vascular invasion are created at the site of the lesion,and must diffuse to the site of the nearest blood vessels (the limbus)in order to exert their effect. Past a certain distance from the limbus,this would no longer be possible and the limbic endothelium would not beinduced to grow into the cornea. Several angiogenic factors are likelyinvolved in this process, many of which are products of the inflammatoryresponse. Indeed, neovascularization of the cornea appears to only occurin association with an inflammatory cell infiltrate, and the degree ofangiogenesis is proportional to the extent of the inflammatory reaction.Corneal edema further facilitates blood vessel ingrowth by loosening thecorneal stromal framework and providing a pathway of “least resistance”through which the capillaries can grow.

Following the initial inflammatory reaction, capillary growth into thecornea proceeds in the same manner as it occurs in other tissues. Thenormally quiescent endothelial cells of the limbic capillaries andvenules are stimulated to divide and migrate. The endothelial cellsproject away from their vessels of origin, digest the surroundingbasement membrane and the tissue through which they will travel, andmigrate towards the source of the angiogenic stimulus. The blind endedsprouts acquire a lumen and then anastomose together to form capillaryloops. The end result is the establishment of a vascular plexus withinthe corneal stroma.

Anti-angiogenic factors and compositions of the present invention areuseful by blocking the stimulatory effects of angiogenesis promoters,reducing endothelial cell division, decreasing endothelial cellmigration, and impairing the activity of the proteolytic enzymessecreted by the endothelium.

Within particularly preferred embodiments of the invention, ananti-angiogenic factor may be prepared for topical administration insaline (combined with any of the preservatives and antimicrobial agentscommonly used in ocular preparations), and administered in eyedrop form.The anti-angiogenic factor solution or suspension may be prepared in itspure form and administered several times daily. Alternatively,anti-angiogenic compositions, prepared as described above, may also beadministered directly to the cornea, Within preferred embodiments, theanti-angiogenic composition is prepared with a muco-adhesive polymerwhich binds to cornea. Within further embodiments, the anti-angiogenicfactors or anti-angiogenic compositions may be utilized as an adjunct toconventional steroid therapy.

Topical therapy may also be useful prophylactically in corneal lesionswhich are known to have a high probability of inducing an angiogenicresponse (such as chemical bums). In these instances the treatment,likely in combination with steroids, may be instituted immediately tohelp prevent subsequent complications.

Within other embodiments, the anti-angiogenic compositions describedabove may be injected directly into the corneal stroma by anophthalmologist under microscopic guidance. The preferred site ofinjection may vary with the morphology of the individual lesion, but thegoal of the administration would be to place the composition at theadvancing front of the vasculature (i.e., interspersed between the bloodvessels and the normal cornea). In most cases this would involveperilimbic corneal injection to “protect” the cornea from the advancingblood vessels. This method may also be utilized shortly after a cornealinsult in order to prophylactically prevent corneal neovascularization.In this situation the material could be injected in the perilimbiccornea interspersed between the corneal lesion and its undesiredpotential limbic blood supply. Such methods may also be utilized in asimilar fashion to prevent capillary invasion of transplanted corneas.In a sustained-release form injections might only be required 2-3 timesper year. Asteroid could also be added to the injection solution toreduce inflammation resulting from the injection itself.

Within another aspect of the present invention, methods are provided fortreating neovascular glaucoma, comprising the step of administering to apatient a therapeutically effective amount of an anti-angiogeniccomposition to the eye, such that the formation of blood vessels isinhibited.

Briefly, neovascular glaucoma is a pathological condition wherein newcapillaries develop in the iris of the eye. The angiogenesis usuallyoriginates from vessels located at the pupillary margin, and progressesacross the root of the iris and into the trabecular meshwork.Fibroblasts and other connective tissue elements are associated with thecapillary growth and a fibrovascular membrane develops which spreadsacross the anterior surface of the iris. Eventually this tissue reachesthe anterior chamber angle where it forms synechiae. These synechiae inturn coalesce, scar, and contract to ultimately close off the anteriorchamber angle. The scar formation prevents adequate drainage of aqueoushumor through the angle and into the trabecular meshwork, resulting inan increase in intraocular pressure that may result in blindness.

Neovascular glaucoma generally occurs as a complication of diseases inwhich retinal ischemia is predominant. In particular, about one third ofthe patients with this disorder have diabetic retinopathy and 28% havecentral retinal vein occlusion. Other causes include chronic retinaldetachment, end-stage glaucoma, carotid artery obstructive disease,retrolental fibroplasia, sickle-cell anemia, intraocular tumors, andcarotid cavernous fistulas. In its early stages, neovascular glaucomamay be diagnosed by high magnification slitlamp biomicroscopy, where itreveals small, dilated, disorganized capillaries (which leakfluorescein) on the surface of the iris. Later gonioscopy demonstratesprogressive obliteration of the anterior chamber angle by fibrovascularbands. While the anterior chamber angle is still open, conservativetherapies may be of assistance. However, once the angle closes surgicalintervention is required in order to alleviate the pressure.

Therefore, within one embodiment of the invention anti-angiogenicfactors (either alone or in an anti-angiogenic composition, as describedabove) may be administered topically to the eye in order to treat earlyforms of neovascular glaucoma.

Within other embodiments of the invention, anti-angiogenic compositionsmay be implanted by injection of the composition into the region of theanterior chamber angle. This provides a sustained localized increase ofanti-angiogenic factor, and prevents blood vessel growth into the area.Implanted or injected anti-angiogenic compositions which are placedbetween the advancing capillaries of the iris and the anterior chamberangle can “defend” the open angle from neovascularization. Ascapillaries will not grow within a significant radius of theanti-angiogenic composition, patency of the angle could be maintained.Within other embodiments, the anti-angiogenic composition may also beplaced in any location such that the anti-angiogenic factor iscontinuously released into the aqueous humor. This would increase theanti-angiogenic factor concentration within the humor, which in turnbathes the surface of the iris and its abnormal capillaries, therebyproviding another mechanism by which to deliver the medication. Thesetherapeutic modalities may also be useful prophylactically and incombination with existing treatments.

Within another aspect of the present invention, methods are provided fortreating proliferative diabetic retinopathy, comprising the step ofadministering to a patient a therapeutically effective amount of ananti-angiogenic composition to the eyes, such that the formation ofblood vessels is inhibited.

Briefly, the pathology of diabetic retinopathy is thought to be similarto that described above for neovascular glaucoma. In particular,background diabetic retinopathy is believed to convert to proliferativediabetic retinopathy under the influence of retinal hypoxia. Generally,neovascular tissue sprouts from the optic nerve (usually within 10 mm ofthe edge), and from the surface of the retina in regions where tissueperfusion is poor. Initially the capillaries grow between the innerlimiting membrane of the retina and the posterior surface of thevitreous. Eventually, the vessels grow into the vitreous and through theinner limiting membrane. As the vitreous contracts, traction is appliedto the vessels, often resulting in shearing of the vessels and blindingof the vitreous due to hemorrhage. Fibrous traction from scarring in theretina may also produce retinal detachment.

The conventional therapy of choice is panretinal photocoagulation todecrease retinal tissue, and thereby decrease retinal oxygen demands.Although initially effective, there is a high relapse rate with newlesions forming in other parts of the retina. Complications of thistherapy include a decrease in peripheral vision of up to 50% ofpatients, mechanical abrasions of the cornea, laser-ihduced cataractformation, acute glaucoma, and stimulation of subretinal neovasculargrowth (which can result in loss of vision). As a result, this procedureis performed only when several risk factors are present, and therisk-benefit ratio is clearly in favor of intervention.

Therefore, within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection of ananti-angiogenic factor(s) (or anti-angiogenic composition) into theaqueous humor or the vitreous, in order to increase the localconcentration of anti-angiogenic factor in the retina. Preferably, thistreatment should be initiated prior to the acquisition of severe diseaserequiring photocoagulation. Within other embodiments of the invention,arteries which feed the neovascular lesions may be embolized (utilizinganti-angiogenic compositions, as described above)

Within another aspect of the present invention, methods are provided fortreating retrolental fibroblasia, comprising the step of administeringto a patient a therapeutically effective amount of an anti-angiogenicfactor (or anti-angiogenic composition) to the eye, such that theformation of blood vessels is inhibited.

Briefly, retrolental fibroblasia is a condition occurring in prematureinfants who receive oxygen therapy. The peripheral retinal vasculature,particularly on the temporal side, does not become fully formed untilthe end of fetal life. Excessive oxygen (even levels which would bephysiologic at term) and the formation of oxygen free radicals arethought to be important by causing damage to the blood vessels of theimmature retina. These vessels constrict, and then become structurallyobliterated on exposure to oxygen. As a result, the peripheral retinafails to vascularize and retinal ischemia ensues. In response to theischemia, neovascularization is induced at the junction of the normaland the ischemic retina.

In 75% of the cases these vessels regress spontaneously. However, in theremaining 25% there is continued capillary growth, contraction of thefibrovascular component, and traction on both the vessels and theretina. This results in vitreous hemorrhage and/or retinal detachmentwhich can lead to blindness. Neovascular angle-closure glaucoma is alsoa complication of this condition.

As it is often impossible to determine which cases will spontaneouslyresolve and which will progress in severity, conventional treatment(i.e., surgery) is generally initiated only in patients with establisheddisease and a well developed pathology. This “wait and see” approachprecludes early intervention, and allows the progression of disease inthe 25% who follow a complicated course. Therefore, within oneembodiment of the invention, topical administration of anti-angiogenicfactors (or anti-angiogenic compositions, as described above) may beaccomplished in infants which are at high risk for developing thiscondition in an attempt to cut down on the incidence of progression ofretrolental fibroplasia. Within other embodiments, intravitreousinjections and/or intraocular implants of an anti-angiogenic compositionmay be utilized. Such methods are particularly preferred in cases ofestablished disease, in order to reduce the need for surgery.

OTHER THERAPEUTIC USES OF ANTI-ANGIOGENIC INFLASTATIN: Anti-angiogenicfactors and compositions of the present invention may be utilized in avariety of additional methods in order to therapeutically treat a canceror tumor. For example, anti-angiogenic factors or compositions describedherein may be formulated for topical delivery, in order to treat cancerssuch as skin cancer, head and neck tumors, breast tumors, and Kaposi'ssarcoma. Within yet other aspects, the anti-angiogenic factors orcompositions provided herein may be utilized to treat superficial formsof bladder cancer by, for example, intravesical administration.

In addition to cancer, however, numerous other non-tumorigenicangiogenesis-dependent diseases which are characterized by the abnormalgrowth of blood vessels may also be treated with the anti-angiogenicfactors or compositions of the present invention. Representativeexamples of such non-tumorigenic angiogenesis-dependent diseases includehypertrophic scars and keloids, proliferative diabetic retinopathy(discussed above), rheumatoid arthritis (discussed above), arteriovenousmalformations (discussed above), atherosclerotic plaques, delayed woundhealing, hemophilic joints, nonunion fractures, Osier-Weber syndrome,psoriasis, pyogenic granuloma, scleroderma, tracoma, menorrhagia(discussed above) and vascular adhesions.

For example, within one aspect of the present invention methods areprovided for treating hypertrophic scars and keloids, comprising thestep of administering one of the above-described anti-angiogeniccompositions to a hypertrophic scar or keloid.

Briefly, healing of wounds and scar formation occurs in three phases:inflammation, proliferation, and maturation. The first phase,inflammation, occurs in response to an injury which is severe enough tobreak the skin. During this phase, which lasts 3 to 4 days, blood andtissue fluid form an adhesive coagulum and fibrinous network whichserves to bind the wound surfaces together. This is then followed by aproliferative phase in which there is ingrowth of capillaries andconnective tissue from the wound edges, and closure of the skin defect.Finally, once capillary and fibroblastic proliferation has ceased, thematuration process begins wherein the scar contracts and becomes lesscellular, less vascular, and appears flat and white. This final phasemay take between 6 and 12 months.

If too much connective tissue is produced and the wound remainspersistently cellular, the scar may become red and raised. If the scarremains within the boundaries of the original wound it is referred to asa hypertrophic scar, but if it extends beyond the original scar and intothe surrounding tissue, the lesion is referred to as a keloid.Hypertrophic scars and keloids are produced during the second and thirdphases of scar formation. Several wounds are particularly prone toexcessive endothelial and fibroblastic proliferation, including bums,open wounds, and infected wounds. With hypertrophic scars, some degreeof maturation occurs and gradual improvement occurs. In the case ofkeloids however, an actual tumor is produced which can become quitelarge. Spontaneous improvement in such cases rarely occurs.

Therefore, within one embodiment of the present invention eitheranti-angiogenic factors alone, or anti-angiogenic compositions asdescribed above, are directly injected into a hypertrophic scar orkeloid, in order to prevent the progression of these lesions. Thefrequency of injections will depend upon the release kinetics of thepolymer used (if present), and the clinical response. This therapy is ofparticular value in the prophylactic treatment of conditions which areknown to result in the development of hypertrophic scars and keloids(e.g., burns), and is preferably initiated after the proliferative phasehas had time to progress (approximately 14 days after the initialinjury), but before hypertrophic scar or keloid development.

As noted above, within yet another aspect of the present invention,vascular grafts are provided comprising a synthetic tube, the surface ofwhich is coated with an anti-angiogenic composition as described above.Briefly, vascular grafts are synthetic tubes, usually made of Dacron orGotrex, inserted surgically to bypass arterial blockages, mostfrequently from the aorta to the femoral, or the femoral to thepopliteal artery. A major problem which particularly complicatesfemoral-popliteal bypass grafts is the formation of a subendothelialscar-like reaction in the blood vessel wall called neointimalhyperplasia, which narrows the lumen within and adjacent to either endof the graft, and which can be progressive. A graft coated with orcontaining anti-angiogenic factors (or anti-angiogenic compositions, asdescribed above) may be utilized to limit the formation of neointimalhyperplasia at either end of the graft. The graft may then be surgicallyplaced by conventional bypass techniques.

Anti-angiogenic compositions of the present invention may also beutilized in a variety of other manners. For example, they may beincorporated into surgical sutures in order to prevent stitchgranulomas, implanted in the uterus (in the same manner as an IUD) forthe treatment of menorrhagia or as a form of female birth control,administered as either a peritoneal lavage fluid or for peritonealimplantation in the treatment of endometriosis, attached to a monoclonalantibody directed against activated endothelial cells as a form ofsystemic chemotherapy, or utilized in diagnostic imaging when attachedto a radioactively labeled monoclonal antibody which recognizesactivated endothelial cells.

Anti-microbial. The pentapeptide of the present invention is alsothought to be an inhibitor of various gram negative and gram positivebacteria and certain viruses, and as such, is suitable at variousdosages as a medical therapeutic in the amelioration of mammaliandiseases in which bacteria or viruses contribute to the pathologicalcondition.

Peptide Production: The peptide of the present invention was firstisolated from the epithelia of a sea cucumber body wall, and can bederived from any species or subphyla of sea cucumber. The peptide can beisolated from the epithelial layer of any sea cucumber, prepared asdescribed in U.S. Pat. No. 5,770,205 by Collin (incorporated herein byreference). It is an object of the present invention to furtherelucidate the anti-cancer activity of sea cucumber tissues. The methodsto prepare isolated sea cucumber epithelial layer are as follows:

The anterior, posterior, viscera and muscles were removed from seacucumber of the species Cucumaria frondosa to obtain an isolated bodywall. Body wall portions thus obtained were heated from about 30 minutesin fresh 170 degree F. water, then cooled on wire racks to roomtemperature. Next, the body wall portions were passed through anindustrial machine known to those in the food processing arts as adeboner or mincer (Paoli Machine, III). The deboner was adjusted toseparate the softer outer epithelial layer from the harder collagenousportion of the body wall. The black viscous layer of the epithelium soseparated, was dried by conventional means using a 40 hp “heat pump”dryer (SouthWind, Nova Scotia, CN) to approximately 5 percent moisturecontent and finely divided to obtain a powder. Any method whereby thesofter epithelial layer is separable from the inner collagenous layer issuitable to produce the raw material from which the peptide of thepresent invention is derived. The epithelial layer can also be keptfrozen and not dried after separation from the skin of any sea cucumber.It is also possible to produce the peptide of the present invention fromsea cucumber body wall which has not had epithelia separated from it,but the percentage of recovery via that method is extremely low and canbe contaminated by different and non-active peptides of the same orsimilar length.

The frozen or dried epithelial layer (1 kg) was ground up and extractedwith one liter of hexane. The hexane was removed and residue thenextracted with one liter of acetone. The acetone and hexane extractswere combined and the solvents were removed by distillation. Theremainder of the material (pulp) was mixed with ½ liter of water and thepH was adjusted to 9.0 using sodium hydroxide (100 ml, 5N), stirred andthen extracted with one liter of ethyl acetate followed by one liter ofnormal butanol. The solvent fractions were combined and taken to drynessat 45 degrees C. with a rotary evaporation with a partial vacuum. Thisfraction contained the peptide of the present invention. The residue wasdissolved in normal butanol, treated with norite and filtered to removea pigment. The material was recrystallized by solvent removal. The yieldof peptide is 60 milligrams from a starting material weight of 1kilogram. The resulting material is a white crystalline material. Theamino acid analysis shows it to be made up of leucine, proline arginine,and serine. Optical rotation C=1, H2O [alpha] 0=125 degrees. Watercontent of one sample was 2 percent. A mass spectroscopy spectrum of thepurified peptide is shown in FIG. 1. The peptides of the presentinvention can also be prepared using standard peptide synthesistechniques known in the art. A mass spectrum of a synthetic preparationof the peptide of SEQ. ID NO. 1 is shown in FIG. 2. An HPLC traceindicating the purity of the preparation appears in FIG. 3.

Accordingly, it is an object of the present invention to provide acompound and method to inhibit unwanted angiogenesis in a human oranimal.

It is yet another object of the present invention to provide acomposition of inhibiting angiogenesis by oral administration of thecomposition.

It is another object of the present invention to provide a treatment fordiseases mediated by angiogenesis.

It is yet another object of the present invention to provide a treatmentfor macular degeneration.

It is yet another object of the present invention to provide a treatmentfor all forms of proliferative vitreoretinopathy including those formsnot associated with diabetes.

It is yet another object of the present invention to provide a treatmentfor solid tumors.

It is yet another object of the present invention to provide a methodand composition for the treatment of blood-born tumors such as leukemia.

It is another object of the present invention to provide a method andcomposition for the treatment of hemangioma.

It is another object of the present invention to provide a method andcomposition for the treatment of retrolental fibroplasia.

It is another object of the present invention to provide a method andcomposition for the treatment of psoriasis.

It is another object of the present invention to provide a method andcomposition for the treatment of Kaposi's sarcoma.

It is another object of the present invention to provide a method andcomposition for the treatment of Crohn's diseases and other inflammatorybowl disease.

It is another object of the present invention to provide a method andcomposition for the treatment of diabetic retinopathy.

Other features and advantages of the invention will be apparent from thefollowing description of preferred embodiments thereof.

EXAMPLE 1 Peptide Production

The peptide of the present invention was first isolated from theepithelia of a sea cucumber body wall, and can be derived from anyspecies or subphyla of sea cucumber. The peptide can be isolated fromthe epithelial layer of any sea cucumber, prepared as described in U.S.Pat. No. 5,770,205 by Collin (incorporated herein by reference). Themethods to prepare isolated sea cucumber epithelial layer are asfollows:

The anterior, posterior, viscera and muscles were removed from seqcucumber of the species Cucumaria frondosa to obtain an isolated bodywall. Body wall portions thus obtained were heated from about 30 minutesin fresh 170 degree F. Water, then cooled on wire racks to roomtemperature. Next, the body wall portions were passed through anindustrial machine known to those in the food processing arts as adeboner or mincer (Paoli Machine, III). The deboner was adjusted toseparate the softer outer epithelial layer from the harder collagenousportion of the body wall. The black viscous layer of the epithelium soseparated, was dried by conventional means using a 40 hp “heat pump”dryer (SouthWind, Nova Scotia, CN) to approximately 5 percent moisturecontent and finely divided to obtain a powder. Any method whereby thesofter epithelial layer is separable from the inner collagenous layer issuitable to produce the raw material from which the peptide of thepresent invention is derived. The epithelial layer can also be keptfrozen and not dried after separation from the skin of any sea cucumber.It is also possible to produce the peptide of the present invention fromsea cucumber body wall which has not had epithelia separated from it,but the percentage of recovery via that method is extremely low and canbe contaminated by different and non-active peptides of the same orsimilar length.

The frozen epithelial layer (1 kg) was ground up and extracted with oneliter of hexane. The hexane was removed and residue then extracted withone liter of acetone. The acetone and hexane extracts were combined andthe solvents were removed by distillation. The remainder of the material(pulp) was mixed with ½ liter of water and the pH was adjusted to 9.0using sodium hydroxide (100 ml, 5N), stirred and then extracted with oneliter of ethyl acetate followed by one liter of normal butanol. Thesolvent fractions were combined and taken to dryness at 45° C. with arotary evaporation with a partial vacuum. This fraction contained thepeptide of the present invention. The residue was dissolved in normalbutanol, treated with norite and filtered to remove pigment. Thematerial was recrystallized by solvent removal. The yield of peptide was60 milligrams from a starting material weight of 1 kilogram. Theresulting material is a white crystalline material.

Amino acid analysis shows the peptide to be made up of leucine, prolinearginine, and serine. Optical rotation C=1, H2O [alpha] 0=125 degrees.Water content of one sample was 2 percent. A mass spectroscopy spectrumof the purified peptide is shown in FIG. 1.

The peptides of the present invention can also be prepared usingstandard peptide synthesis techniques known in the art. A mass spectrumof a synthetic preparation of the peptide of SEQ. ID NO. 1 is shown inFIG. 2. An HPLC trace indicating the purity of the preparation appearsin FIG. 3.

EXAMPLE 2 In Vivo Anti-Inflammatory Activity

The anti-inflammatory activity of the isolated peptide of Example 1 wastested by oral administration to rats in an Adjuvant Induced Arthritisassay and by a Mouse Ear Edema assay, as are known in the arts.

Adjuvant Induced Arthritis Model: Male Sprague-Dawley rats (160-180 g)were sensitized by injecting Fruend's Complete Adjuvant (0.5% suspensionof killed mycobacterium tuberculosis (H37RA, Difco in mineral oil)).0.05 ml was administered intradermally at a plantar site on the righthind leg of each rat. The test materials were given orally (by gavage)in 0.5% methylcelluose, at a dose level of 20 milligrams per kilo bodyweight, and given once per day for 14 days. The administration wasstarted the day after sensitization. The left hind paw was measured justbefore sensitization and on Day 14, and the plantar edema inhibitoryrate and the body weight gain rate were determined in comparison withthe nonsensitized rat groups. The paw weights for each group in eachtest run were averaged. Activity was calculated as follows:${\frac{{Mean}\quad {paw}\quad {weights}\quad {of}\quad {controls}\text{-}{Mean}\quad {paw}\quad {weights}\quad {of}\quad {test}\quad {group}}{{Mean}\quad {paw}\quad {weights}\quad {of}\quad {test}\quad {group}} \times 100} = {\% \quad {anti}\quad \text{-}{inflammatory}\quad {response}}$

The Anti-inflammatory response is the difference in the mean footvolume. This is also calculated as a “percent inhibition.”Phenylbutazone and hydrocortisone assayed concurrently with InflaStatinand the same 20 mg per KG body weight gave responses of 112.2% and132.6% respectively while the peptide of the present invention gave ananti-inflammatory response of 116.3%. FIG. 4.

Mouse-ear edema model. Croton-oil, which contains a variety of phorbolesters, and arachidonic acid (AA) are standard inducers of inflammationin mouse ears when applied topically. Phorbol esters, especially phorbolmyristyl acetate (PMA), activate one or more isozymes of the ubiquitousmembrane receptor, protein kinase C (PKC), which in turn initiatesseveral metabolic cascades leading to inflammation. As is a substratefor one of these cascades, leading to prostaglandin and leukotrieneproduction, both of which are inflammatory. Inhibition of suchinflammation by a putative bioactive agent is considered a generalizedindicator of potential pharmacologic activity.

Male Swiss Webster mice (15-20 g) were purchased from B&K Universal,Kent, Wash. and maintained in the laboratory for one day prior toentering the experiment. At t=0, the ventral side of both ears of eachmouse was challenged topically with 10 λ of 10% croton oil in acetone.At t=30 min, the same pinnal area was treated topically with 20 λ ofeither carbopol (n=2 ears) or 5% peptide dissolved in carbopol (n=4ears). The animals were sacrificed by cervical dislocation at t=24 hrs,the ears removed, and a biopsy take from each ear with an 8 mm punch.The biopsy was immediately weighed on a 4-place balance. Results showedthat the peptide of the present invention produced a 61% reduction inswelling compared to vehicle-only treated ears.

EXAMPLES 3-9 In Vitro Activities

A variety of activities of the peptide of Example 1 were tested in arange of in vitro assays.

I. MATERIALS AND EQUIPMENT

A. Test Substances and Dosing Pattern

Peptide was prepared according to the methods set out in Example 1. Thevehicle of distilled water was used for in vitro assays, workingsolution of peptide was added to the 10 ml tissue bath. For in vivoassay, distilled water and saline was used as vehicle in oral andintraperitoneal administration, respectively. Dosing volume was 20 ml/kgfor mice or 10 ml/kg for rats. For topical route, peptide was dissolvedin a vehicle of Acetone/Ethanol (1:1) and applied in a dosing volume 20μl/ear.

B. Chemicals

Aluminum Hydroxide (Sigma, U.S.A.), Acetone (Wako, Japan), BSA (BovineSerum Albumin), CaCl₂ (Merck, Germany), Croton Oil (Sigma, U.S.A.),Cyclophosphamide Monohydrate (Sigma, U.S.A.), DNBS(2,4-Dinitrobenzenesulfonic Acid, TCI, Japan), Dimethyl Sulfoxide (Mark,Germany), Ethanol (Merck, Germany), Galanin (Sigma, U.S.A.), (+)Glucose(Sigma, U.S.A.), Histamine-diphosphate (Sigma, U.S.A.), Indomethacin(Sigma, U.S.A.), KCI (Wako, Japan), KH₂PO₄ (Wako, Japan), MgSO₄7H₂O(Wako, Japan), Mepyramine Maleate (Sigma, U.S.A.), NaHCO₃ (Merck,Germany), NaCl (Merck, Germany), Ovalbumin (Sigma, U.S.A.), Oxazolone(Sigma, U.S.A.), PAF (Sigma, U.S.A.), Pentobarbital Sodium (Tokyo Kasei,Japan), Propranolol-HCl (Sigma, U.S.A.), Pyrogen-Free Saline (Astar,Taiwan), Pyrilamine Maleate (Sigma, U.S.A.), R-α-Methyl histamine (RBI,U.S.A.), Succinylcholine Chloride (Asta-Werke, Bielefeld, Germany),Sulfasalazine (Sigma, U.S.A.), WEB 2086 (Boehringer Ingelheim). Dosesfor compounds in salt form were calculated on the basis of the weight ofthe salt.

C. Equipment

Dyer model micrometer gauge (Peacock, Japan), EKG: Cardionec (NECSan-ei, Japan), Isotonic transducer # 50-6360 (Harvard, U.S.A.), 2-penrecorder SS-250F (Sekonic, Japan), Micropipet # P20, P200 and P1000(Gilson, U.S.A.), Mouse scale Z-40 (Taconic), Pen oscillograph Type 8K(NEC San-ei, Japan), Pressure transducer # P23×L (Viggo-Spectramed,U.S.A.), Pneumatic pulse transducer (Narco, U.S.A.), Rodent ventilator #683 (Harvard, U.S.A.), Recorder # SS-250 (Sekonic, Japan), Transduceramplifier # 50-7970 (Harvard, U.S.A.).

D. Animals

In these studies, male/female Wistar derived rats, male Sprague-Dawleyderived rats, male/female Duncan Hartley derived guinea pigs and maleICR derived mice provided by MDS Panlabs Taiwan, Ltd. were used. Spaceallocation for animals was as follows: 45×23×15 cm for 10 mice, 45×23×15cm for 6 rats and 43×21×20 cm for 3 guinea pigs. The animals were housedin APECR (Allentown Gaging, Allentown, N.J. 08501, U.S.A.) cages. Freeaccess to standard lab chow (Fwusow Industry Limited Co., Taiwan) andtap water was granted. All animals were maintained in a controlledtemperature (22°-24° C.) and humidity (60%-80%) environment with 12 hourlight dark cycles for at least one week in Panlabs laboratory prior touse. All aspects of this work including housing, experimentation an ddisposal of animals were performed in general according to theInternational Guiding Principles for Biomedical Research InvolvingAnimals (CIOMS Publication No. ISBN 92 90360194, 1985).

Total Tested Animals: 45 mice and 9 rats and 9 guinea pigs

Total Tested Tissues: 4

II. METHODS AND RESULTS EXAMPLE 3 Galanin Agonism/Antaonism

A segment of ileum obtained from Wistar derived male or female ratsweighing 275±25 gms and sacrificed by CO₂ overexposure was used. Thetissue was placed under 1 g tension in a 10 ml bath containing Kreb'ssolution pH 7.4 at 32° C. Test substance (30 μM)-induced contraction 50percent or more (≧50%) within 5 minutes, relative to control 0.1 μMgalanin response, indicated possible galanin receptor agonist activity.Atha test substance concentration where no significant agonist activitywas seen, ability to reduce the galanin-induced contractile response byso percent or more (≧50%) indicated galanin receptor antagonist activity.Each concentration was tested on two separate preparations. The resultsare set forth below in Table 1.

TABLE 1 Galanin Agonism/Antagonism Compound Route Conc. N AgonismAntagonism Vehicle in vitro 0.1 ml 1 0 0 (Distilled Water) in vitro 0.1ml 1 0 0 Ave. 0 0 Peptide in vitro 30 μM 1 0 24 in vitro 30 μM 1 0 0Ave. 0 12 Galanin in vitro 0.1 μM 1 100 ND in vitro 0.1 μM 1 100 ND Ave.100 ND A segment of rat ileum was placed under 1 g tension in a 10 mlbath Krebs solution pH 7.4 at 32° C. Test substance-induced isotonicallyrecorded contraction within 5 minutes indicated agonist activity. Theability to reduce the 0.1 μM galanin-induced contractile responseindicated antagonist activity. ND: Not determined because agonistactivity was significant. Total tested tissues in this assay: 2

EXAMPLE 4 Histamine H₃ Agonism/Antagonism

A segment of ileum obtained from Duncan Hartley derived male or femaleguinea pigs weighing 325±25 gms and sacrificed by CO₂ overexposure wereused. The tissue was placed under 0.5 g tension in a 10 ml bathcontaining Kreb's solution pH 7.4 and pyrilamine (0.3 μM) at 32° C. andsubjected to field stimulation (70% of maximum voltage, 0.1 Hz, 0.5millisecond). Test substance (30 μM)-induced reduction of isometricallyrecorded contractions 50 percent or more (≧50%) within 5 minutes,relative to control 0.3 μM R-α-methylhistamine response, indicatedpossible histamine H₃ receptor agonist activity. At a test substanceconcentration where no significant agonist activity was seen, ability toinhibit R-α-methylhistamine-induced response by 50 percent or more(≧50%) indicated histamine H₃ receptor antagonist activity. Eachconcentration was tested on two separate preparations. The results areset forth below in Table 2.

TABLE 2 Histamine H₃ Agonism/Antagonism Antag- Compound Route Conc. NAgonism onism Vehicle in vitro 0.1 ml 1 0 0 (Distilled Water) in vitro0.1 ml 1 0 0 Ave. 0 0 Peptide in vitro 30 μM 1 4 0 in vitro 30 μM 1 0 7Ave. 4 4 R (α)-Methylhistamine in vitro 0.3 μM 1 100 ND in vitro 0.1 μM1 100 ND Ave. 100 ND Thioperamide in vitro 0.3 μM 1 0 90 in vitro 0.3 μM1 0 96 Ave. 0 93 A segment of guinea pig ileum was placed under 0.5 gtension and subjected to field stimulation in a 10 ml bath Krebssolution pH 7.4 at 32° C. Test substance-induced isometrically recordedrelaxation within 5 minutes indicated agonist activity. The ability toreduce the 0.3 μM R(α)-methylhistamine-induced relaxation responseindicated antagonist activity. ND: Not determined because agonistactivity was significant. Total tested tissues in this assay: 2

EXAMPLE 5 Immune Suppression, Cellular

Groups of 5 ICR derived male mice weighing 22±1 gms were used. Thepreshaved abdominal surface was sensitized by application of oxazolone0.1 ml of 5% solution. Test substance of peptide at dose of 100 mg/kgand vehicle (saline) were administered into the mice intra peritoneallyafter one hour and daily for five consecutive doses. After additionalfour days, the animals were challenged by secondary application ofoxazolone (0.025 ml of 5% solution) to the right ear. After a further 24hours, each mouse was sacrificed and ear thickness measured with a DyerModel micrometer gauge. A 30 percent or more (≧30%) decrease relative tothe vehicle control group was considered significant and indicatedpossible immunosuppressant activity. The results are set forth in Table3, below.

TABLE 3 Immune, Suppression, Cellular Thickness (× 0.1 mm) R. L. %Compound Route Dose N Ear Ear Net INH Vehicle- IP 20 ml/kg × 5 1 52 2032 control IP 20 ml/kg × 5 2 53 22 31 (Saline) IP 20 ml/kg × 5 3 47 2225 IP 20 ml/kg × 5 4 45 22 23 IP 20 ml/kg × 5 5 50 23 27 X 27.6  0 SEM1.7 Peptide IP 100 mg/kg × 5 1 43 23 20 IP 100 mg/kg × 5 2 43 22 21 IP100 mg/kg × 5 3 44 20 24 IP 100 mg/kg × 5 4 Died Died Died IP 100 mg/kg× 5 5 50 23 27 X 23.0 17 SEM 1.6 Cyclo- IP 30 mg/kg × 5 1 38 22 16phosphamide IP 30 mg/kg × 5 2 26 16 10 IP 30 mg/kg × 5 3 32 18 14 IP 30mg/kg × 5 4 23 16 7 IP 30 mg/kg × 5 5 40 20 20 X 13.4 51 SEM 2.3 Testcompound was administered IP in groups of 5 mice one hour aftersensitization and daily for five consecutive doses. The challenge ofoxazolone was applied on day 8 and the ear thickness was recorded 24hours later. One animal out of 5 tested animals died on day 8 beforeoxazolone application. Total tested animals in this assay: 15 mice

EXAMPLE 6 Inflammation, Inflammatory Bowel Disease

Groups of 3 Sprague-Dawley derived male rats weighing 150±20 gms andfasted for 24 hours were used. Distal colitis was induced byintra-colonic instillation of DNBS (2,4-dinitrobenzene sulfonic acid, 30mg in 0.5 ml ethanol 30%) after which air (2 ml) was gently injectedthrough the cannula to ensure that the solution remains in the colon.Test substance was administered PO (30 mg/kg) at 24 and 2 hours beforeDNBS instillation. Then, the animals received test compound every 24hours for 5 consecutive days. The control group was given vehicle aloneas compound dosing pattern. The animals were sacrificed 24 hours afterthe final dose of test compound administration and each colon wasremoved and weighed. Colon-to-body weight ratio was obtained from thepercentage of the comparison between the animal colon weight and bodyweight on the 8^(th) day. A 30 percent or more (±30%) reduction incolon-to-body weight ratio relative to the vehicle treated control groupwas considered significant. The results are set forth in Table 4, below.

TABLE 4 Inflammation, Inflammatory Bowel Disease Ratio Compound RouteDose No. Ind. Net % INH. Blank-control — — 1 0.406 — — 2 0.353 — — 30.352 X 0.370 — — SEM 0.018 Vehicle-control PO 10 ml/kg × 7 1 0.672(Distilled Water) PO 10 ml/kg × 7 2 0.768 PO 10 ml/kg × 7 3 0.736 X0.725 0.355 0 SEM 0.028 Peptide PO 30 mg/kg × 7 1 0.683 PO 30 mg/kg × 62 Died PO 30 mg/kg × 7 3 0.608 X 0.646 0.276 22 SEM 0.038 SulfasalazinePO 300 mg/kg × 7 1 0.496 PO 300 mg/kg × 7 2 0.508 PO 300 mg/kg × 7 30.650 X 0.551 0.181 49 SEM 0.050 Test substance was administered PO (30mg/kg) at 24 and 2 hours before DNBS instillation and then daily for 5days. Groups of 3 tested rats were sacrificed hours after the finalcompound administration. The colon-to-body weight ratio was recorded.Ratio: Colon (g)/8^(th) B.W. × 100%. One out of 7 tested animals died onday 7 after daily compound administration. Total tested animals in thisassay: 9 rats

EXAMPLE 7 Platelet Activating Factor (PAF) Antagonism

Groups of 5 male ICR mice weighing 22±2 gms were employed. At dose of100 mg/kg test substance dissolved in a vehicle of distilled water wereadministered orally. The control group received vehicle alone. At 60minutes after dosing, the animals were injected intravenouslyPAF-acether (100 μ/kg IV, dissolved in 0.25% BSA). One hour later, thesurvival animals were recorded. The prevention of PAF-induced mortalityin 50 percent or more (≧50%) of mice indicated significant activity. Theresults are set forth in Table 5, below.

TABLE 5 Platelet Activating Factor (PAF) Antagonism No. of CompoundRoute Dose N Survival % Protection Vehicle (2% Tween 80) PO  20 ml/kg 50 0 Peptide PO 100 mg/kg 5 0 0 WEB-2086 PO  3 mg/kg 5 3 60 Test compoundwas administered PO (100 mg/kg) to a group of 5 mice at one hour beforeinjection of PAF-acether (100 μg/kg IV), then the number of survival wasrecorded. Total tested animals in this assay: 15 mice

EXAMPLE 8 Pulmonary, Histamine H₃

A group of Duncan Hartley derived male or female guinea pigs weighing250±50 gms were anesthetized with pentobarbital sodium (50 mg/kg IP plusan additional 15 mg/kg IP if required) and succinylcholine chloride (2mg/animal IP) was subsequently administered to prevent spontaneousrespiration. Body temperature was maintained at 37° to 38° C. Thetrachea was cannulated and the guinea pig ventilated with a Harvardrodent respirator in a closed system. Tracheal pressure (TP) wasrecorded through a side-arm of the cannula connected to a P23ID Stathamtransducer. Respiratory rate set at 50 strokes/minute with a strokevolume (approximately 1 ml/100 g) sufficient to produce a baselinetracheal pressure of 6 cm H₂O. Mean arterial pressure was monitored froma cannulated carotid artery, and heart rate was obtained from chestelectrodes arranged for lead II. The jugular vein was cannulated for IVvehicle or drug administration in a volume of 1 ml/kg. Guinea pigs weresensitized with IP injections of ovalbumin 0.5 μg+Al(OH)₃ 1 mg (0.5ml/animal) on days 1, and boosted with same dosage of ovalbumin andAl(OH)₃ vaccine on day 8; and the animals were ready to be challengedbetween days 19 and 23 to get a submaximal ovalbumin (50 μg/kgIV)-induced bronchoconstriction, reflected as an increase in trachealpressure (cm H₂O). The animals were pretreated 5 minutes before testsubstance administration with IV indomethacin (10 mg/kg), mepyramine (2mg/kg), and propranolol (0.1 mg/kg): a “cocktail” designed to inhibitthe generation of cyclooxygenase products (thromboxanes, etc.) as wellas antagonize histamine and β-adrenergic receptors. Test substances wereadministered PO (10 mg/kg) at 60 minutes before ovalbumin (50 μg/kg IV)challenge in 3 guinea pigs. Tracheal pressure, blood pressure and heartrate were measured immediately before and after ovalbumin challenge. A50 percent or more (≧50%) inhibition of the induced bronchoconstrictionrelative to vehicle treated control animals was considered significant.The results are set forth in Tables 6-1 and 6-2, below.

TABLE 6-1 Pulmonary, Histamine H₃ Inc. of TP (cm H₂O) Compound RouteDose N Ind. X ± SEM % INH. Vehicle PO 5 ml/kg 1 28.0 (Distilled PO 5ml.kg 2 20.0 Water) PO 5 ml/kg 3 40.5 29.5 ± 6.0 0 Peptide PO 10 mg/kg 127.5 PO 10 mg/kg 2 35.5 PO 10 mg/kg 3 31.5 31.5 ± 2.3 −6.8 R-α- IV 30mg/kg 1 8.5 methylhistamine IV 30 mg/kg 2 25.0 IV 30 mg/kg 3 0.5 11.3 ±7.2 61.6 A group of 3 guinea pigs were sensitized with IP injections ofovalbumin 0.5 μg + Al(OH)₃ 1 mg (0.5 ml/animal) on days 1 and day 8 andready to be challenged between days 19 and 23. The animals wereanesthetized with pentobarbital sodium and body temperature wasmaintained at 37° to 38° C. Test substances were administered PO (10mg/kg) at 60 minutes before ovalbumin # (50 μg/kg IV) challenge.Tracheal pressure, blood pressure and heart rate were measuredimmediately before and after ovalbumin challenge. A 50 percent or more(≧50%) inhibition of the induced bronchoconstriction relative to vehicletreated control animals was considered significant.

TABLE 6-2 Pulmonary, Histamine H₃ Pre- Post- Compound Route Dose Nchallenge challenge Blood Pressure (mmHg) Vehicle (Saline) PO  5 ml/kg 359.3 ± 4.1 44.0 ± 4.2 Peptide PO 10 mg/kg 3 60.7 ± 5.2 44.7 ± 0.7R-α-methylhistamine IV 30 mg/kg 3 55.3 ± 1.3 34.0 ± 4.2 Heart Rate(beats/min) Vehicle (Saline) PO  5 ml/kg 3  228 ± 6.9   184 ± 10.6Peptide PO 10 mg/kg 3  224 ± 4.0   188 ± 14.4 R-α-methylhistamine IV 30mg/kg 3  232 ± 4.0 216 ± 0.0  A group of 3 guinea pigs were sensitizedwith IP injections of ovalbumin 0.5 μg + Al(OH)₃ 1 mg (0.5 ml/animal) ondays 1 and day 8 and ready to be challenged between days 19 and 23. Theanimals were anesthetized with pentobarbital sodium and body temperaturewas maintained at 37° TO 38° C. Test substances were administered PO (10mg/kg) at 60 minutes before ovalbumin (50 μg/kg # IV challenge. Trachealpressure, blood pressure and heart rate were measured immediately beforeand after ovalbumin challenge. A 50 percent or more (≧50%) inhibition ofthe induced bronchoconstriction relative to vehicle treated controlanimals was considered significant. Total tested animals in this assay:9 guinea pigs

EXAMPLE 9 In Vitro Screening of Peptide Activities

The peptide was further screened in vitro in a variety of enzyme andbiochemical assays. Methods employed were adapted from the scientificliterature to maximize reliability and reproducibility. The primaryliterature reference for each assay is listed in Table 11 opposite theresults for each assay.

Unless otherwise indicated, the peptide was tested in duplicate at theconcentrations indicated for each assay. Results presented in Table 10,“Results,” are the average of duplicate determinations, unless otherwisenoted.

Reference compounds were tested concurrently at 5 concentrations as anintegral part of each assay to ensure the validity of the resultsobtained. Data inclusion criteria required that the IC₅₀ of theconcurrently tested reference compound fall between ⅓ (0.33×) and 3times (3×) the historical IC₅₀ of the reference compound (shown in Table9, “Reference Compound Data”).

Enzyme Assays

Enzyme assays were performed under conditions described in Table 8,“Experimental Conditions.”. The commercial suppliers and catalog numbersof substrates employed in this study are given in Table 7, “MaterialSources.” The name, commercial source, and catalog numbers of concurrentreference compounds (controls) employed in this study are given in Table9, “Reference Compound Data.” The reaction catalyzed by each enzyme islisted in Table 8, “Experimental Conditions.”

Radioligand Binding Assays

Radioligand binding assays were performed under conditions described inTable 8, “Experimental Conditions.” Radioligands employed in this studyare given in Table 8, and commercial sources and catalog numbers of theradioligands are provided in Table 7, “Material Sources.” Unlabeled,blocking ligands employed this study are listed in Table 8, “ExperimenalConditions,” along with the commercial sources and catalog number of theunlabeled ligand. The concurrent reference compound (control) employedin each assay is listed in Table 9, “Reference Compound Data,” alongwith the commercial supplier and catalog number of the compound. Sourcesof receptors or membrane preparations, whether from animal tissue orfrom recombinant expression, are provided in Table 7 as well. Wherereceptors have been obtained through recombinant expression techniques,the species from which the cDNA was obtained, and the type of expressionsystem employed (mammalian, insect, bacterial) is also given Table 7.

Maximum total binding and nonspecific binding were determined each timeeach assay was run. Nonspecific binding was defined as the proportion oftotal binding not displaced by unlabeled ligand specific for thereceptor. Specific binding was defined as the proportion of totalbinding that was displaced by unlabeled ligand. The unlabeled ligand andconcentration employed are given Table 8, “Experimental Conditions.”

Where presented, IC₅₀ values (defined as the concentration of testcompound or competing ligand capable of displacing 50% of the specificbinding of the radioligand) were determined by a non-linear, leastsquares regression analysis using GraphPad Prism Software (GraphPad, SanDiego, Calif., USA). Where inhibition constants (K_(i)) are presented,the K_(i) values were calculated using the equation of Cheng and Prusoff(Cheng, Y., Prusoff, W. H., Biochem. Pharmacol. 2:3099-3108, 1973) usingthe observed IC₅₀ of the tested compound, the concentration ofradioligand employed in the assay, and the historical values for theK_(d) of the ligand (obtained experimentally). Where presented, the HillCoefficient (n_(H)), defining the scope of the competitive bindingcurve, was calculated using GraphPad Prism. Hill coefficientssignificantly different than 1.0, may suggest that the bindingdisplacement does not follow the laws of mass action with a singlebinding site. Where IC₅₀, K_(i), and/or Hill Slope data is presentedwithout Standard Error of the Mean (SEM), data are insufficient to bequantitative, and the values presented (K_(i), IC₅₀, nH) should beinterpreted with caution.

Biochemical assay results are presented in Table 10, as the percentinhibition of specific binding or activity, and unless noted are theaverage of duplicate tubes tested at each concentration.

TABLE 7 MATERIAL SOURCES Material Rad.Ligand/ Target Source SubstrateSolvent Collagenase IV human U937 NEN, Cat. 0.4% DMSO cells #NET-660Cyclooxygenase-1 ram seminal Sigma, Cat #A-4425 1% DMSO vesicleCyclooxygenase-2 sheep placenta Sigma, Cat. 1% DMSO #A-44255-Lipoxygenase rat RBL-1 cells Sigma, Cat. 1% DMSO #A-9673 PLA₂,Pancreatic porcine Amersham, Cat. 1% DMSO pancreas #CFA-656 Protease -Elastase human Sigma, Cat. 1% DMSO neutrophils #M4765 Ca++/Calmodulinrat brain Panlabs, U.S. 1% DMSO Dep. PK II Protein Kinase C, rat brainSigma, Cat #H-5505 1% DMSO Non-Select. Protein Kinase rabbit brainSigma, Cat. 1% DMSO C - γ (recombinant) #H-5505 Ca++ Ch.-L, rat cerebralNEN, Cat. 0.4 DMSO Benzothiazepine cortex #NET-847 Ca++ Ch.-L, ratcerebral NEN, Cat 0.4% DMSO Dihydropyridine cortex #NET-741 Histamine H₃rat brain NEN, Cat. 0.4% DMSO #NET-1027 Interleukin-1α mouse Swiss/Amersham, Cat. 0.4% DMSO 3T3 cells #IM-205 Interleukin-6 human U266 NEN,Cat. 0.4% DMSO cells #NEX-269 Leukotriene B₄ human U937 Amersham, Cat.0.4% DMSO cells #TRK-692 TNF-α human U937 NEN, Cat. 0.4% DMSO cells#NEX-257

TABLE 8 EXPERIMENTAL CONDITIONS ‘Reaction or ‘Nonspecific Cat. # AssayTarget *Ligand/Substrate Ligand Time/Temp 11400 Collagenase IV[²H]Denatured Collagen [³H]Collagen-[³H]Peptides 90 min. @ 37° C. 11600Cyclooxygenase-1 Arachidonic Acid AA-PGG₂-PGH₂-PGE₂ 20 min. @ 37° C.11800 Cyclooxygenase-2 Arachidonic Acid AA-PGG₂-PGH₂-PGE₂ 20 min. @ 37°C. 13600 5-Lipoxygenase Arachidonic Acid AA-5-HPETE-5-HETE 8 min. @ 25°C. 16000 PLA₂ Pancreatic [¹⁴c]3-phosphatidyl Phosphatidylcholine- 5 min.@ choline Palmitate 37° C. 16600 Protease - Elastase MeOSuc-AAPV-pNaSubstrate-MeOSuc-AAPV + pNA 10 min. @ 25° C. 16800 Ca++/Calmodulin Dep.PK BB40 BB40 + [γ³²P]ATP-[³²P]BB40 + 5 min. II ADP @ 30° C. 17800Protein Kinase C, Non- Histone H1 (HH1) HH1 +[γ³²P]ATP-[^(32 P]HH1 + ADP) 15 min. @ Select. 25° C. 18400 ProteinKinase C - γ Histone H1 (HH1) HH1 + [γ³²P]ATP-[³²P]HH1 + ADP 5 min. @30° C. 21450 Ca++ Ch.-L, [³H]Diltiazem 10 μM Diltiazem; Kali- 60 min. @Benzothiazepine Chemie 37° C. 21460 Ca++Ch.-L, [³H]Nitrendipine 1 μMNifedipine; Sigma, 90 min. @ Dihydropyridine Cat. #N-7634 25° C. 23980Histamine H. [³]NAMH 1 μM NAMH; Sigma, Cat. #M- 30 min. @ 4910 22° C.24350 Interleukin-1α [²⁵I]IL-1 alpha 50 nM interleukin-1α; Pepro 120min. @ Tech EC, Cat. #200-01A 37° C. 24410 Interleukin-6[¹²⁵I]Interleukin-6 40 nM Interleukin-6; Pepro 16 hr. @ 4° C. Tech EC,Cat. #200-06 25051 Leukotriene B₄ [²⁵H]LTB₄ 2 μM Leukotriene B₄; Sigma,30 min. @ Cat. #L-0517 25° C. 28650 THF-α [125I]TNF-alpha 50 nM TNF-α; R& D, Cat 3 hr. @ 4° C. #210-TA *Radioligand or Enzyme Substrate, ¹EnzymeAssays Only, ²Unlabeled blocking ligand used for Radioligand BindingAssays Only, §Criteria or brief description given for tissue, animal,and anti-infective assays

TABLE 9 REFERENCE COMPOUND DATA Cat. # Target ^(1K) ₂ IB. #Spec. Ref.Cmpd. Supplier ¹IC, ¹K₁ 11400 Collagenase IV HS—CH—R— Peptides 31 nMCH(CH— Int., CH(CH₃)₂—CO— Cat. Nal-Ala-NH #ISN- 3835-PI 11600Cyclooxygenase-1 Indomethacin Sigma, 1.7 μM Cat. #I- 7378 11800Cyclooxygenase-2 Indomethacin Sigma, 2.4 μM Cat. #I7378 136005-Lipoxygenase NDGA Sigma, 0.26 Cat #N- μM 5023 16000 PLA₂ PancreaticQuinacrine Sigma, 120 μM Cat. #Q3251 16600 Protease - N-Me—OSuc- Sigma,180 nM Elastase AAPV Cat. #M- 0398 16800 Ca++/Calmodulin TrifluoperazineSigma. 27 μM Dep. PK II Cat. #T- 8516 17800 Protein Kinase H-7 Sigma, 35μM C, Non-Select Cat. #I- 7016 18400 Protein Kinase C StaurosporineSigma, 2.4 nM Cat. #S- 4400 21450 Ca++Ch.-L, 30 nM 330 80% DiltiazemKall- 25 nM 23 nM Benzothiazepine fmol/mg Cheml 21460 Ca++ Ch.-L, 0.18230 91% Nifedipine Sigma, 2.7 nM 1.7 nM Dihydropyridine nM fmol/mg Cat.#N- 7634 23980 Histamine H₂ 0.35 13 85% NAMH Sigma, 2.9 nM 0.75 nMfmol/mg Cat. #M- nM 4910 24350 Interleukin-1α 0.18 4100 80% IL-1 alphaPepro 31 pM 23 pM nM R/cell Tech EC, Cat. #200-01A 24410 Interleukin-60.06 670 80% IL-6 Pepro EC, 32 nMM 1.4 nM nM R/cell Cat. #200-06 25051Leukotriene B 0.07 78 85% LTB₄ Sigma, 0.2 nM 0.05 nM fmol/mg Cat. #L- nM0517 28650 TNF-α 37 pM 11 65% TNF-alpha R&D, Cat. 84 pM 32 pM pmole/mg#210-TA Historical values obtained are shown for each protocol,¹Historical K₁, B_(max) and % Specific Binding are shown for radioligandbinding assays and were experimentally determined by saturationanalysis. Historical reference ligand K₁ values shown for binding assaysonly.

TABLE 10 RESULTS Target ¹Spp. Conc. ¹% Inhibition References CollagenaseIV Hum 100.0 μM 2 Morodomi, T., Ogata, Y., Sasaguri, Y., Morimatsu, M.,Nagase, H. (1992) Purification and characterization of matrixmetalloproteinase 9 from U937 moncytic leukaemia and HT 1080fibrosarcoma cells. Biochem. J. Kato, Y., Ogawa, K., Yamamoto, S., Abe,S., Kishi, J., and Hayakawa, T. (1990) A novel TIMP-insensitive type IVcollagen-degrading metalloproteinase from murine metastatic sarcoma.FEBS Lett. 268:39-42. Mallya, S.K., Mookhtiar, D.A., and Van Wart, H.E.(1986) Accurate, quantitative assays for the hydrolysis of soluble typeI, II and III ³H-acetylated collagens by bacterial and tissuecollagenases. Anal. Biochem. 158:334345, 1986. Cyclo- Ram 300.0 μM 2Boopathy, R. and Balasubramanian, oxygenase-1 A.S. (1988) Purificationand characterization of sheep and platelet cyclooxygenase. Biochem. J239:371-377. Evans, A.T., Formukong, E.A., and Evans, F.J. (1987) Actionof cannabis constituents on enzymes of arachidonate metabolism:Anti-inflammatory potential. Biochem. Pharmacol. 36:2035-2037. Cyclo- Ov300.0 μM 2 O'Sullivan, M.G., Huggins, E.M. oxygenase-2 Jr., Meade, E.A.,DeWitt, D.L., McCall, C.E. (1992) Lipopolysaccharide inducesprostagladin H synthase-2 in alveolar macrophages. Biochem. Biophys.Res. Commun. 187:1123- 1127. Evans, A.T. , Formukong, E.A., and Evans,F.J. (1987) Action of cannabis constituents on enzymes of arachidonatemetabolism: Anti-inflammatory potential. Biochem. Phamacol.36:2035-2037. Boopathy, R. and Balasubramanian, A.S. (1988) Purificationand characterization of sheep platelet cyclooxygenase. Biochem. J.239:371-377. 5-Lipoxygenase Rat 30.0 μM 0 Egan, R.W. and Gale, P.H.(1985) Inhibition of mammalian 5- lipoxygenase by aromatic disulfides.J. Biol. Chem. 260:11554-11559. Shimizu, T., Radmark, O., andSamuelsson, B. (1984) Enzyme with dual lipoxygenase activities catalyzesleukotriene A₄ synthetase from arachidonic acid. Proc. Natl. Acad. Sci.USA 81:689-693. PLA₂, Por 300.0 μM −11 Katsumata, M., Gupta, C.,Pancreatic Goldman, A.S. (1986) A rapid assay for activity ofphospholipase A₂ using radioactive substrate. Anal. Biochem.154:676-681. Protease - Hum 30.0 μM −11 Baugh, R.J. and Travis, J.(1976) Elastase Human leukocyte granule elastase: rapid isolation andcharacterization. Biochemistry 14:836-841, 1976. Ca++/Calmodulin Rat100.0 μM 12 Lai, Y., Nairn, A.C., Greengard, Dep. PK II P. (1986)Autophosphorylation reversibly regulates the Ca²⁺/calmodulin- dependenceof Ca²⁺/calmodulin dependent protein kinase II. Proc. Natl. Acad. Sci.USA 83:4253-4257. Protein Kinase Rat 300.0 μM −6 Hannum, Y.A., Loomis,C.R., Bell, C₁ Non-Select. R.M. (1985) Activation of protein kinase C byTriton-X-100 mixed micelles containing diacylglycerol andphosphatidylserine. J. Biol. Chem. 260:10039-10043. Jeng, A.Y., Sharkey,N.A. and Blumberg, P.M. (1986) Purification of stable protein kinase Cfrom mouse brain cytosol by specific ligand elution using fast proteinliquid chromatography. Cancer res. 46:1966-1971. Protein Kinase Rab100.0 μM −6 Woddgett, J.R., and Hunter, T. C - γ (1987) Isolation andcharacterization of two distinct forms of protein kinase C.J. Biol.Chem. 262:4836- 4843. Ca++ Ch.-L, Rat 10.0 μM 18 Schoemaker, H. andLanger S.Z. Benzothiazepine (1985) [³H]Diltiazem binding to calciumchannel antagonist recognition sites in rat cerebral cortex. Eur. J.Pharmacol. 111:273-277. Ca++ Ch.-L, Rat 10.0 μM 2 Gould R.J., Murphy,K.M.M., Dihydro- Snyder, S.H. (1982) pyridine [³H]nitrendipine-labeledcalcium channels discriminate inorganic calcium agonists andantagonists. Proc. Natl. Acad. Sci. USA 79:3656-3650. Ehlert, F.J.,Roeske, W.R., Itoga, E., and Yamamura, H.I. (1982) The binding of[³H]nitrendipine to receptors for calcium channel antagonists in theheart, cerebral cortex and ileum of rats. Life Sci. 30:2191-2202.Histamine H₃ Rat 10.0 μM −13 Korte, A., Myers, J., Shih, N.Y., Egan,R.W., Clark, M.A. (1990) Characterization and tissue distribution of H₃histamine receptors in guinea pigs by N- alpha-methylhistamine. Biochem.Biophys. Res. Commun. 168:979- 986. West Jr., R.E., Zweig, A., Shih,N.Y. Seigel, M.I., Egan, R.W., and Clark, M.A. (1990) Identification oftwo H₃- histamine receptor subtypes. Mol. Pharmacol. 38:610-613.Interleukin-1α Mus 10.0 μM 15 Bird, T.A. and Saklatvaia J. (1986)Identification of a common class of high-affinity receptors for bothtypes of porcine interleukin-1 on connective tissue cells. Nature324:263- 266. Chin, J., Cameron, P.M., Rupp, E., and Schmidt, J.A.(1987) Identification of a high affinity receptor for nativeinterleukin-1 α and interleukin-1β on normal human lung fibroblasts. J.Exp. Med. 165:70-86. Killian, P.L. Kaffka, K.L. Stern, A.S., Woehle, D.,Benjamin, W.R. Dechiara, T.M. Gubler, U., Farrar, J.J. Mizel, S.B., andLomedico, P.T. (1986) Interleukin-1α and interleukin-1β bind to the samereceptors on T cells. J. Immunol. 136:4509- 4514. Interleukin-6 Hum 10.0μM 13 Taga, T., Hibi, M., Hirata, Y., Yamasaki, K., Yasukawa, K.Matsuda, T., Hirano, T., Kishimoto, T. (1989) Interleukin-6 triggers theassociation of its receptor with a possible signal transducer, gp 130.Cell 58:573-581. Cornfield, L.J., and Sills, M.A. (1991) High affinityinterleukin-6 binding sites in bovine hypothalamus. Eur. J. Pharmacol.202:113-115. Leukotriene B₄ Hum 10.0 μM 8 Winkler, J.D., Sarau, H.M.Foley, J.J. Mong, S. Crooke, S.T. (1988) Leukotriene B₄-inducedhomologous desensitization of calcium mobilization and phosphoinositidemetabolism in U-937 cells. J. Pharmacol. Exp. Ther. 246:204- 210. TNF-αHum 10.0 μM −11 Yoshie, O., Tada, K., and Ishida, N. (1986) Binding andcrosslinking of ¹²⁵I labeled recombinant human tumor necrosis factor tocell surface receptors. J. Biochem. 100:531- 541. Maloff, B.L. andDelmendo, R.E. (1991) Development of a high-throughput binding assay forinterleukin- 1α(IL-1α) and tumor necrosis factor (TNF-α) in isolatedmembrane preparations. Agents and Actions 34:32-34. Negative valuescorrespond to stimulation of binding or enzyme activity ¹Bac = Bacteria;Bov = Bovine; Chi = Chicken; GP = Guinea Pig; Hum = Human; Mus = Mouse;Ov = Ovine; Por = Pig; Rab = Rabbit; SyH = Syrian Hamster; Yea-Yeast

EXAMPLE 10 In Vivo Anticancer Activity

The peptide was assayed in a mouse model of the tumor Sarcoma 180. Thismodel has been used extensively as indicative of neovascularization inmammals inasmuch as the tumor so implanted is quickly vascularizing.

Experimental Design: All mice were weighed at test initiation and testtermination. The animals were divided into three groups. The Sarcoma 180tumor was made up from a passage mouse to furnish 2×1 viable cells per0.1 mL inoculum. The cells were injected into the left leg hamstringmuscle mass. The was delivered to the mice by I.P. injections, or gavageon days 1-5.

At the termination of the experiments, the mice were weighed and theleft rear leg was amputated at the thigh. The skin was removed from theleg to expose the site at which the tumor was located. The net tumorweight was determined by subtracting the mean value obtained from 10normal legs. The results are shown below in Table 11.

TABLE 11 Anti-Cancer Activity Against Mouse Tumor Sarcoma 180 Test Meantumor % tumor Material No. of mice Wt. gain, gm wt. gain, gm inhibitionControl, 10 6.2 2.3 — water natural 6 5.8 0.03 99.0 peptide @ 10 mg/kgsynthetic 6 5.9 1.45 58.6 peptide (crude 60%) @ 10 mg/kg

EXAMPLE 11 CAM Assay

The peptide was assayed in the CAM assay, so called, and inhibitedproliferation of vacularization in that model by approximately 95%.Method: The method used was that described in D. Knighton, D. Ausprunk,D. Tapper, and J. Folkman, “Avascular and Vascular Phases of TumourGrowth in the Chick Embryo.” J. Cancer 35:347-355 (1977) Procedure: Thetest compounds were suspended in sterile saline and then applied tomethylcellulose discs, ¼ inch diameter with a micropipette and allowedto air dry at a concentration of 10 μg/disc.

The test was graded as follows:

0 No change from control embryos +1 Slight inhibition of vasculature +2Moderate inhibition of vasculature +3 Almost complete inhibition ofvasculature +4 Complete inhibition of vasculature

Results: The CAM assay antiangiogenesis scores are summarized in thetable below. The following scores are based on the observation of atleast 10 chick embryos.

The results were as follows:

Saline blank (neg. control) 0.12 Protamine/Hydrocortisone 2.7 (positivecontrol) Peptide 3.6

ADDITIONAL REFERENCES

1. Tatemoto, K., Rokaeus, A., Jornvall, H., McDonald, T. J., and Mutt,V. Galanin-A novel biologically active peptide from porcine intestine.FEBS Lett. 164: 124-128, 1983.

2. Hew, R. W. S., Hodgkinson, C. R., and Hill, S. J. Characterization ofhistamine H₃-receptor in guinea pig ileum with H₃-selective ligands. Br.J. Pharmacol. 101: 621-624, 1990.

3. Griswold, D. E., DiLorenzo, J. A. and Calabresi, P. Quantificationand pharmacological dissection of oxazolone-induced contact sensitivityin the mouse. Cellular Immunology 11:198-204, 1974.

4. Hogaboam, C. M., Muller, M. J., Collins, S. M. and Hunt, R. H. Anorally active non-selective endothelin receptor antagonist, bosentan,markedly reduces injury in a rat model of colitis. Eur. J. Pharmacol.309: 261-269, 1996.

5. Chang, J., Blazek, Skowronek, M. Mariniari, L. and Carlson, R. P. Theantiinflammatory action of guanabenz is mediated through 5-lipoxygenaseand cyclooxygenase inhibition. Eur. J. Pharmacol. 142: 197-205, 1987.

6. Motaslm Billah, M., Chapman, R. W., Egan, R. W., Gilchrest, H.,Piwinski, J. J., Sherwood, J., Siegel, M. I., West, Jr., R. E. andKreutner, W. SCH 37370: a potent, orally active dual antagonist ofplatelet activating factor and histamine. J. Pharmacol. Exp. Ther. 252:1090-1096, 1990.

7. Konzett, H. And Rossler, R. Versuensanordnung zu Untersuchungen ander Bronchialmuskulatur. Arch. Exp. Pathol. 195: 71-74, 1940.

8. Danko, G., Hey, J. A., Egan, R. W., Kretner, W. And Chapman, R. W.Histamine H₃ receptors inhibit sympathetic modulation of airwaymicrovascular leakage in allergic guinea pigs. Eur. J. Pharmacol. 254:283-286, 1994.

3 1 5 PRT Homo sapiens 1 Leu Pro Pro Ser Arg 1 5 2 5 PRT ArtificialSequence Generic Formula 2 Xaa Xaa Xaa Xaa Xaa 1 5 3 5 PRT ArtificialSequence Generic Formula 3 Xaa Pro Pro Xaa Xaa 1 5

I claim:
 1. A method for inhibiting an inflammatory response in a mammalin need thereof comprising administering to said mammal an effectiveamount of a peptide of the formula Leu-Pro-Pro-Ser-Arg (SEQ. ID 1). 2.The method of claim 1, wherein said mammal suffers from InflammatoryBowel Disease.
 3. The method of claim 2, wherein said mammal suffersfrom Crohn's disease.
 4. The method of claim 1, wherein said peptide isadministered by injection.
 5. The method of claim 1, wherein saidpeptide is administered orally.
 6. The method of claim 1, wherein saidpeptide is administered topically.
 7. The method of claim 1, whereinsaid mammal is a human.
 8. The method of claim 1, wherein said peptideis administered in a dose of about 2.5 mg/kg to about 500 mg/kg.
 9. Themethod of claim 1, wherein said peptide is administered topically in adose of 5 mg/kg to 100 mg/kg.
 10. The method of claim 1, wherein saidpeptide is administered in a dose of 30 mg/kg.